Terminal cell differentiation is accompanied by permanent withdrawal from the cell division cycle. The molecular mechanisms that couple these two processes are poorly understood. Progression of eukaryotic cells through the cell cycle is primarily regulated by a family of structurally related serine/threonine protein kinases, which consists of a regulatory subunit, a cyclin, and a catalytic subunit, a CDK (cyclin dependent kinase). The enzymatic activity of the CDK subunit is regulated by cyclin binding and activation, subunit phosphorylation, and association with CDK inhibitors. The ability of CDK inhibitors to arrest the cell cycle and their induction in response to a variety of growth inhibitory signals, including growth suppression by both the pRb and p53 tumor suppressors, present the CDK inhibitors as a group of ideal molecules that may link cell cycle arrest to cell differentiation. Using the 3T3-L1 preadipocyte cell line as a model system, the subunit composition of CDK complexes, CDK kinase activity, and cell cycle progression during adipocyte differentiation will be analyzed. The function of two CDK inhibitors, p18 and p21, in causing and/or maintaining cell cycle arrest during adipocyte differentiation, as suggested by preliminary experiments, will be tested directly using antisense RNA technology. The involvement of the Rb protein family and p53 in regulating terminal adipocyte differentiation and their interaction with CDK inhibitor pathways will be determined using the HPV16-E6 and HPV16-E7 oncoproteins and dominant negative mutants of pRb and p53.